A field investigation of Bacillus anthracis contamination of U.S. Department of Agriculture and other Washington, D.C., buildings during the anthrax attack of October 2001

Abstract

In response to a bioterrorism attack in the Washington, D.C., area in October 2001, a mobile laboratory (ML) was set up in the city to conduct rapid molecular tests on environmental samples for the presence of Bacillus anthracis spores and to route samples for further culture analysis. The ML contained class I laminar-flow hoods, a portable autoclave, two portable real-time PCR devices (Ruggedized Advanced Pathogen Identification Device [RAPID]), and miscellaneous supplies and equipment to process samples. Envelopes and swab and air samples collected from 30 locations in the metropolitan area once every three days were subjected to visual examination and DNA extraction, followed by real-time PCR using freeze-dried, fluorescent-probe-based reagents. Surface swabs and air samples were also cultured for B. anthracis at the National Veterinary Service Laboratory (NVSL) in Ames, Iowa. From 24 October 2001 to 15 September 2002, 2,092 pieces of mail were examined, 405 real-time PCR assays were performed (comprising 4,639 samples), and at the NVSL 6,275 samples were subjected to over 18,000 platings. None of the PCR assays on DNA extracted from swab and air samples were positive, but viable spores were cultured from surface swabs taken from six locations in the metropolitan area in October, November, and December 2001 and February, March, and May 2002. DNA extracted from these suspected B. anthracis colonies was positive by real-time and conventional PCRs for the lethal factor, pXO1, and for capA and vrr genes; sequence analysis of the latter amplicons indicated >99% homology with the Ames, vollum, B6273-93, C93022281, and W-21 strains of B. anthracis, suggesting they arose from cross-contamination during the attack through the mail. The RAPID-based PCR analysis provided fast confirmation of suspect colonies from an overnight incubation on agar plates.

FIG. 1.

(A) Results of real-time B. anthracis LF PCR performed with the RAPID instrument using DNA extracted from a bacterial colony with morphology suggestive of B. anthracis, cultured from a swab sample taken from the Economic Research Service (ERS2) mail room in Washington, D.C., October 2001. Relative fluorescence is plotted on the y axis, and cycle number is plotted on the x axis. The plot for the positive control is indicated; the horizontal plots represent negative controls and non-B. anthracis samples. (B) Results of real-time PCR for the pXO1 locus performed on the Smart Cycler on DNA extracted from suspected B. anthracis colonies cultured from samples taken at the Economic Research Service (ERS2), the Forest Service (Frst Srvc) (November 2001), and the USDA South Building (S Bldg) (Washington, D.C., November 2001). The Sterne strain positive control (Sterne), no template control (ntc), and extraction control (G3) are indicated. (C) Results of real-time B. anthracis LF PCR performed with the RAPID instrument using DNA extracted from swabs spiked with 1,000 and 100 spores of B. anthracis Sterne. Plots for the positive control (+ctrl) and the 1,000- and 100-spore-spiked samples are indicated; horizontal plots represent negative samples (these include other replicates spiked with 1,000, 100, 10, and 1 spores, and extraction and no template controls).

FIG. 2.

Results of capA gene PCR (6) performed on DNA extracted from swabs and bacterial colonies suspected of harboring or being B. anthracis. Lane L, DNA ladder with rung sizes indicated; lanes 6B and 11B, DNAs extracted from swab samples; lane 1, DNA extracted from a colony cultured from the USDA South Building mail room, Washington, D.C., November, 2001; lane 2, DNA extracted from a colony cultured from the Forest Service mail room, November 2001; lane 3, DNA extraction control; lane 4, B. anthracis Pasteur strain positive control; lane ntc, no template control.